highly permeable polylactide-caprolactone nerve guides enhance peripheral nerve regeneration through...

Biomaterials 20 (1999) 1489}1500

Highly permeable polylactide-caprolactone nerve guides enhanceperipheral nerve regeneration through long gaps

Francisco J. RodrmHguez!, Nuria GoH mez!, Gabriele Perego", Xavier Navarro!,*!Department of Cell Biology and Physiology, Universitat Auto% noma de Barcelona, E-08193 Bellaterra, Spain

"Novamont SpA, I-28100 Novara, Italy

Received 21 September 1998; accepted 20 March 1999

Abstract

We compared regeneration and functional reinnervation after sciatic nerve resection and tubulization repair with bioresorbableguides of poly(L-lactide-co-e-caprolactone) (PLC) and permanent guides of polysulfone (POS) with di!erent degrees of permeability,leaving a 6 mm gap in di!erent groups of mice. Functional reinnervation was assessed to determine recovery of motor, sensoryand sweating functions in the hindpaw during four months postoperation. Highly permeable PLC guides allowed for faster andhigher levels of reinnervation for the four functions tested than impermeable or low-permeable PLC guides, while semipermeable 30and 100 kDa POS tubes yielded very low levels of reinnervation. The regeneration success rate was higher with PLC than withPOS tubes. Morphometrical analysis of cross-sectional nerves under light microscopy showed the highest number of regeneratedmyelinated "bers at mid tube and distal nerve in high-permeable PLC guides. Impermeable PLC guides allowed slightly worselevels of regeneration, while low-permeable PLC guides promoted neuroma and limited distal regeneration. The lowest numberof regenerated "bers were found in POS tubes. In summary, highly permeable bioresorbable PLC guides o!er a suitable alterna-tive for repairing long gaps in injured nerves, approaching the success of autologous nerve grafts. ( 1999 Elsevier Science Ltd.All rights reserved.

Keywords: Nerve regeneration; Tube repair; Reinnervation; Morphometry; Bioresorbable guide; Permeable guide

1. Introduction

The usual method of repair of a transected nerveinvolves mobilization and epineurial suturing of theproximal and distal stumps with coaptation of individualnerve fascicles. When a nerve gap is incurred that cannotbe repaired by end-to-end suture without tension, thecurrent repair method is a sutured autologous graft fromanother nerve of lesser functional importance. The useof autologous grafts has some disadvantages such asthe need of a second surgical step, loss of the donornerve function, a limited supply of donor nerves and themismatch between nerve and graft dimensions. An alter-native repair method is tubulization, which involvesenclosure of the ends of a severed nerve by a tube whichholds the stumps in place, o!ers a guide to regenerating

*Corresponding author. Tel.: #34-3-581-1966; fax: #34-3-581-2986.E-mail address: [email protected] (X. Navarro)

axons to the distal stump, and may concentrate neuro-trophic products from the nerve stumps [1}3]. The use ofsynthetic tubes as nerve guides to bridge a nerve gap hasprovided an excellent in vivo experimental model tostudy the process of peripheral nerve regeneration. Syn-thetic silicone tubes have been used in a clinical trial incomparison with direct suture or short autografts torepair sectioned nerves in the man forearm with success-ful results [4]. However, the main objection for usingpermanent tubes is that they will remain in situ after thenerve has regenerated and may cause a chronic foreignbody reaction and late nerve compression with second-ary complaints and impairment of nerve function [5,6].Nerve guides made of biodegradable materials mightovercome these problems if, after allowing the outgrowthand maturation of the nerve, they are gradually degradedwithout signi"cant deformation. Several types of bio-resorbable nerve conduits have been shown to o!er anadequate guide for nerve regeneration and may consti-tute an acceptable substitute for the use of an autologousnerve graft to repair short or medium length gaps in

0142-9612/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 0 5 5 - 1

peripheral nerves [7}9], but less is known about theircapabilities to allow regeneration over long gaps.

The degree of permeability of the nerve guide may alsoin#uence nerve regeneration. It has been postulated thatsemipermeable conduits may enhance nerve regenerationover impermeable ones [10}12], although in other re-ports these "ndings were not corroborated [13}15]. Thefavorable e!ects of permeable tubes may be attributed todi!erent reasons such as: metabolic exchange across thetube wall, di!usion into the guide lumen of growth pro-moting factors generated in the external environment,retention of trophic factors secreted by the nerve stumps,or a combination of all these [12]. Hence, the size of thetube wall porus and its stability over time seem to beimportant factors to determine the #ow of di!erentconstituents that may promote or inhibit regeneration.However, little is known about the combined e!ects ofpermeable and resorbable nerve guides. In this study weevaluated, by means of functional and histologicalmethods, axonal regeneration and target organs reinner-vation after resection of the mouse sciatic nerve leavinga 6 mm gap and repair with bioresorbable and durabletubes of di!erent degrees of permeability. The mainobjective was to assess the e!ectiveness of new permeableresorbable nerve guides made of poly(L-lactide-co-e-cap-rolactone).

2. Materials and methods

2.1. Surgical procedure

Operations were performed under pentobarbital anes-thesia (60 mg/kg i.p.) on "ve groups of female Swiss OF1mice, aged 2.5 months. First, the saphenous nerve wascut in the femoral space and a long segment of the distalstump removed to prevent regeneration. The sciaticnerve was then exposed at the midthigh, transected ata constant point, 45 mm from the tip of the third digit,and a segment of the distal stump resected. The lesionwas repaired by "xing the nerve stumps 1 mm inside theends of a tube, by means of one 10-0 suture stitch at eachend, leaving an interstump gap of 6 mm (Fig. 1). Micewere divided into "ve groups according to the tubes usedfor repair:

f Group PLC-np (n"9): bioresorbable non-permeabletubes of poly(L-lactide-co-e-caprolactone) (PLC).

f Group PLC-hp (n"6): bioresorbable tubes of PLC ofhigh permeability.

f Group PLC-lp (n"12): bioresorbable tubes of PLC oflow permeability.

f Group POS-30 (n"7): durable tubes of polysulfonewith a MW cut-o! of 30 kDa (Amicon).

f Group POS-100 (n"6): durable tubes of polysulfonewith a MW cut-o! of 100 kDa (Amicon).

All PLC tubes were of 1 mm i.d. and 150 lm thick-ness, while POS tubes had 1.1 mm i.d. and 250 lmthickness.

Once implanted, the tubes were "lled with physiolo-gical saline solution. Finally, the skin was closed with 5-0silk sutures and disinfected with povidone}iodine solu-tion. In order to avoid autotomy after denervation, ani-mals were pretreated with amitriptyline in the drinkingwater [16]. The experimental protocols followed the rec-ommendations of the European Union for the care anduse of laboratory animals and were approved by theEthics Committee of our institution.

2.2. Characteristics of the nerve guides

The PLC tubes were prepared as previously described[17]. Brie#y, 6 g of the synthesized poly(L-lactide-co-e-caprolactone) 50 : 50 by weight were dissolvedin 100 ml of anhydrous ethyl acetate. A glass mandrel,1 mm in diameter, lubricated with glycerin and rotat-ing at 30 rpm was immersed in the solution. It wasthen slowly pulled out in order to obtain a uniformcoating. The mandrel was immersed in cold, anhydrousmethanol for 4 min and, still rotating, the solventwas evaporated under a mild stream of dry nitrogen.After "ve successive coatings, the tube was stripped o!the mandrel and immersed in anhydrous methanol for24 h, then dried at 253C and 0.5 mm Hg for a further24 h.

For the preparation of PLC nerve guides of highpermeability the solution of poly(L-lactide-co-e-caprolac-tone) was "lled with a "ne powder of glucose, accuratelyground to get about 10 lm particles. The glucose, inamount equal to that of the copolymer, was thoroughlysuspended, then the suspension was used in the same wayas previously described to prepare nerve guides. Waterextraction for 24 h at room temperature was su$cient tocompletely free the samples from the "ller, as gravimetri-cally determined. However, in order to prevent any mor-phological change of the samples during storage, thepreparation of the nerve guides was terminated withoutany extraction of glucose, that takes place directly invivo. As determined by UV spectroscopy, proteins withmolecular weight of 240 kDa (catalase) and of 450 kDa(b-galactosidase), as well as 2000 kDa dextran blue, caneasily di!use at room temperature during a 24 h periodthrough the pores that are generated in the nerve guidewall with water extraction.

The preparation of permeable PLC nerve guides of lowpermeability was performed by "lling the solution ofpoly(L-lactide-co-e-caprolactone) with a "ne powder ofamylose, made of particles of less than 10 lm. Theamylose, in amount equal to that of the copolymer, wasaccurately suspended, then the suspension was used inthe same way as previously described. The hygroscopicbehavior of amylose, together with long resorption times,

1490 F.J. Rodrn&guez et al. / Biomaterials 20 (1999) 1489}1500

Fig. 1. Photographs showing (A) the sciatic nerve repaired with a highly permeable poly(L-lactide-co-e-caprolactone) (PLC) guide at the time ofimplantation, and (B) the appearance of a nerve regenerated through a PLC guide. The proximal stump is at the top of each picture. The length of theguide is 8 mm.

o!ered an intermediate situation between those of nonpermeable and highly permeable nerve guides. ThePLC-lp guides have initially a low permeability (below240 kDa from the protein di!usion tests performed) thatwill slowly increase in a time-dependent mode as thestarch is slowly extracted in water and the biodegrada-tion advances. Under scanning electron microscopy, afterextraction of the corresponding saccaride, the permeablePLC guides showed a smooth, although slightly irregularsurface with porus of variable size homogeneouslytraversing their wall (Fig. 2).

The POS tubes feature a partially fenestrated outerlayer and a semipermeable inner layer connected by anopen trabecular network which provides the guide'sstructural support. The inner layer has a molecularweight cut-o! of 30 and 100 kDa in the tubes used, andshows a very smooth internal surface (Fig. 3).

2.3. Functional tests

Regeneration of large myelinated nerve "bers was as-sessed by nerve conduction studies [18]. The sciaticnerve was stimulated percutaneously through a pair ofneedle electrodes at the sciatic notch, and the compoundmuscle action potential (CMAP) recorded from plantarand gastrocnemius muscles with microneedle electrodes,while for sensory nerve conduction, the electrodes wereinserted on the fourth toe to record the compound nerveaction potentials (CNAP) of the digital nerves. Squarepulses of 0.01 ms duration were applied with increasingamplitude up to about 25% above the voltage thatevoked a maximal response. The evoked CMAP andCNAP were displayed on a storage oscilloscope (Tek-tronix 2221) at settings appropriate to measure the am-plitude from baseline to the maximal negative peak and

F.J. Rodrn&guez et al. / Biomaterials 20 (1999) 1489}1500 1491

Fig. 2. SEM micrographs of poly(L-lactide-co-e-caprolactone) guides of high (A, C, E) and low (B, D, F) permeability. (A, B) View of the guide internalsurface (bar"75 lm). (C, D) Closer view of the internal side (bar"20 lm). (E, F) Transversal section through the guide wall (bar"86 lm).

the latency from stimulus to the onset of the "rst negativede#ection. The animal temperature was controlled bya water circulating heating pad.

Reinnervation by small-size nerve "bers was evaluatedby testing sympathetic sudomotor and nociceptive re-sponses. Sweating was stimulated by injection of pilocar-pine nitrate (5 mg/kg s.c.). Ten minutes later a siliconematerial (Elasticon, Kerr Co.) was applied over the plan-tar surface of the hindpaw [18,19]. As the materialhardened, it retained the impressions made by the sweatdroplets emerging from individual sweat glands (SGs).The number of SG impressions was determined undera dissecting microscope with transillumination. Recoveryof pain sensitivity was tested by light pricking witha needle, under a dissecting microscope, at "ve areas,

from the most proximal pawpad to the tip of the seconddigit on the plantar aspect of the denervated paw [18].A subjective score to pinprick (PP) was assigned from noresponse (0), reduced or inconsistent responses (1) tonormal reaction (2) in each area tested, in comparisonwith the responses to the same stimuli applied to thecontralateral intact hindpaw.

Functional tests were performed before operation toobtain baseline control values and at several intervals upto four months postoperation. For normalization, valuesobtained after operation were expressed as the percent-age of preoperative values for each mouse, and plottedagainst time. For all functions tested we calculated theday of the "rst response after denervation (when therewas no reinnervation an arbitrary value of 150 days was


Fig. 3. SEM micrographs of polysulfone guides. (A, B) View of a 30 kDa polysulfone guide internal surface (bar"750 lm in A and 86 lm in B).(C) Transversal section through a 30 kDa guide wall and (D) a 100 kDa guide wall (bars"120 lm). Note the thin smooth inner layer that conferssemipermeability and the thick outer trabecular layer.

assigned), the percentage of maximal recovery achievedduring follow-up (with zero values for mice without rein-nervation), and an overall functional recovery index(FRI) representing the area under the reinnervation curve[18].

2.4. Histological studies

At the end of functional follow-up, animals were re-anesthetized, the operated nerve was dissected from sur-rounding tissues, and a long segment including theimplanted guide removed and "xed in glutaral-dehyde}paraformaldehyde (3}3%) in 0.1 M cacodylatebu!er (pH 7.4, 4 h, 43C). The nerves were cut in twopieces to enable transverse sections at the mid tube andat the distal nerve. Samples were post"xed in OsO

4(2%,

2 h), dehydrated through ethanol series, and embedded inEpon. Transverse semithin sections (0.5 lm) of the wholenerve were stained with toluidine blue and examinedunder light microscopy. Microphotographs were made at"nal magni"cation of 200] for measuring the cross-

sectional area of the whole nerve, and of 2000] formorphometrical analysis. Morphometry was done from7}10 randomly selected "elds containing at least 500myelinated "bers. If the whole nerve section had less than500 "bers, all the regenerated "bers were measured. Thetotal number of myelinated "bers in the nerve was esti-mated from the area occupied by the "bers in the photo-graphs. Images were redrawn on a digitizing tablet andprocessed through a Macintosh computer using a mor-phometry software to obtain axon and "ber perimeters.Areas and diameters of axons and "bers, as well as theg ratio (ratio of axon to "ber perimeter) were calculated.When there was no nerve regeneration at the cross-sectional level, zero values were entered for nerve areaand number of myelinated "bers, but the sample was notused for morphometrical measurements.

All data are expressed as mean$SEM. Statisticalcomparisons between groups were made by non-para-metric Kruskal-Wallis and Mann-Whitney U tests, andChi-squared test for the proportion of success. The di!er-ences were considered signi"cant when P(0.05.


Fig. 4. Evolution of (A) the number of reactive sweat glands (SG) and(B) the pinprick (PP) score over time in groups of mice with sciatic nerveresection and repair with di!erent nerve guides made of poly(L-lactide-co-e-caprolactone) (PLC) or polysulfone (POS).

3. Results

All animals recovered uneventfully from surgery. Dur-ing follow-up, six mice of group PLC-lp developed severeautotomy of late onset (15}20 days) and had to be eutha-nized by one month postsurgery. At "nal inspection theguides were swollen and showed longitudinal cracks,there was a neuroma formed at the proximal stump andno nerve tissue regenerated inside the tube lumen in anyof them. These mice are therefore excluded from furtheranalysis.

3.1. Functional reinnervation

Sudomotor nerve regeneration, judged by the re-appearance of secreting SGs over time, is shown inFig. 4A. After denervation, the number of reactive SGsdecreased within two weeks to 0 or only a few inconsist-ently reactive. The "rst reinnervated SGs were foundaround 40 days postoperation (dpo) in groups repairedwith high permeable and non-permeable PLC guides,and signi"cantly later in the other three groups (Table 1).The number of reactive SGs increased progressively toachieve a maximal recovery of 66% of control values ingroup PLC-hp, 53% in group PLC-np, and 46% ingroup PLC-lp, while groups repaired with POS tubesshowed steady low responses during follow-up (P(0.05vs. groups PLC-hp and PLC-np).

The earliest nociceptive responses were found at prox-imal sites of the hindpaw between 20 and 30 dpo in somemice of group PLC-hp (Fig. 4B), increasing with a highslope to a maximal response of 67% by 50 dpo. In theother groups, withdrawal responses to pinprick were "rstobserved by 40 dpo in group PLC-np, 50 dpo in groupPLC-lp, and 60 dpo in groups POS-30 and POS-100.The total PP score increased slowly to 41% of controls ingroup PLC-np, 51% in group PLC-lp and 24% ingroups POS-30 and POS-100.

The "rst low-amplitude CMAPs were recorded ingastrocnemius muscles by 30 dpo in group PLC-hp,40 dpo in group PLC-np, 50 dpo in PLC-lp, and 90 dpoin groups POS-30 and POS-100 (Fig. 5A). The meanCMAP amplitude showed a slow increase during follow-up, achieving levels of recovery of 56% in group PLC-hp,32% in group PLC-np, 28% in PLC-lp and less than10% in groups POS-30 and POS-100 with respect topreoperative values. Reinnervation of plantar musclesstarted slightly later and followed a similar course(Fig. 5B), although the mean CMAP amplitudes reachedlower levels than in the proximal gastrocnemius muscle,27% in group PLC-hp, 15% in group PLC-np, 9% inPLC-lp and less than 1% in groups POS-30 and POS-100 with respect to preoperative values. The latency ofthe CMAPs shortened to reach steady values that were inall cases longer than normal, and signi"cantly more pro-longed in group POS-100 than in the other groups.

The digital CNAPs were recorded in a few animalsfrom 50 dpo in group PLC-hp, from 60 dpo in groupPLC-np, and from 105 dpo in group PLC-lp (Fig. 5C).Their amplitude increased throughout the study in groupPLC-hp to reach a maximal recovery of 33%, whilegroups PLC-np and PLC-lp showed a slight increase tomaximal responses of about 10%. No responses wereelicited in mice with POS guides.

The group repaired with a high-permeable PLC guideshowed a shorter mean onset of reinnervation and anoverall FRI 2}12 times higher than the other groups(Table 1). The FRI of group PLC-np was twice that ofgroup PLC-lp for the large myelinated "bers but similarfor thin "bers reinnervation. Reinnervation was very lowin mice repaired with POS tubes, mainly for motor andsensory functions mediated by large "bers. Di!erenceswere not signi"cant in some between groups compari-sons due to the large within group variability, because ofa proportion of animals did not show any degree ofrecovery during follow-up. The proportion of mice withe!ective functional recovery was 66% in groups PLC-hpand PLC-lp, 55% in group PLC-np and around 30% ingroups POS-30 and POS-100 (Table 1).

3.2. Morphological regeneration

During microscopic dissection the guides were foundcovered by a well vascularized "brous tissue, thinner in


Table 1Reinnervation success rate, onset day of reinnervation and functional recovery index (FRI) for each function tested and the average during four monthsfollow-up after resection and tube repair leaving a 6 mm gap in the "ve groups of mice studied. Values are expressed as mean$SEM

Group PLC-np PLC-lp PLC-hp POS-30 POS-100

n 9 6# 6 7 6Reinnervation 5 (55%) 4 (66%) 4 (66%) 2 (29%) 2 (33%)

Onset daySG 73$11 87$20 63$19 126$9!," 145$5!,"

PP 102$16 94$19 69$26 129$14 130$15G-CMAP 92$16 92$20 74$24 124$17 120$15P-CMAP 105$15 108$18 76$24 135$9 145$5!

CNAP 130$11 135$9 89$19 150$0! 150$0!

Mean 103$12 107$18 74$22 134$9!," 142$6!,"

FRISG 2766$772 2021$670 4564$1306 532$263!," 10$10!,"

PP 2208$1006 2435$1005 5730$1814 625$404 1098$940G-CMAP 1334$559 800$346 2675$1005 145$100 153$86P-CMAP 666$406 205$150 1195$408 7$5!," 4$4!,"

CNAP 311$175 151$109 1231$485 0$0!," 0$0!,"

Mean 1457$568 1136$516 3079$970 286$136!," 253$201!,"

P(0.05.!vs. group PLC-hp."vs. group PLC-np.#Failure of regeneration in other six mice, excluded because of autotomy.

the PLC-hp and PLC-np tubes than in the other tubesimplanted. PLC tubes appeared softened and only partlyresorbed (Fig. 1B). In regenerated cases pinching thedistal nerves evoked a re#ex response in the anesthetizedanimals, while pinching and cutting the "brous cover ofthe tube did not, indicating that axonal regeneration hadprogressed inside the tube. A newly formed nerve cablewas present inside the guides of successfully regeneratedcases.

Sections taken at the midtube demonstrated typicalregenerated nerve cables surrounded by loose connectivetissue, and composed of a central core of myelinated andunmyelinated axons arranged in small fascicles, as well as"broblasts, Schwann cells and blood vessels (Fig. 6). Forcomparisons, all animals were included in measurementsof the regenerated nerve area and the number of myel-inated "bers (Table 2). The area of the intratubular regen-erated cable at midtube ranged between 0.01 and0.14 mm2, being in all cases smaller than the normalmouse sciatic nerve (0.21 mm2). The mean cross-sectionalarea was higher in group PLC-hp than in the othergroups studied. The mean number of myelinated "berswere higher within and distal to PLC-hp tubes than inthe other tubes, although di!erences did not attainstatistical signi"cance. While similar numbers of re-generated myelinated "bers are found in the tube and inthe distal nerve in groups PLC-np and PLC-hp, therewas only a low proportion of axons that reached thedistal nerve in groups PLC-lp, POS-30 and POS-100. Inparticular, there were high numbers of myelinated axons

(more than 7000) in two mice of group PLC-lp at the tubelevel, but only a few (less than 300) distally, indicating thegeneration of intratubular neuroma.

Quantitative morphometrical analysis was performedonly in mice with a regenerated cable inside the chamber(Table 2). The mean diameter of myelinated axonsat midtube and distal nerve levels was smaller thanin control nerves. The smallest values were found ingroup PLC-lp. The g ratio was closer to normal nerves inthe regenerated nerves of group PLC-hp than in theother groups, indicating a better myelin to axonal caliberratio.

4. Discussion

The practical interest of research on arti"cial guidesfor nerve regeneration is mainly the achievement of analternative to the autologous nerve graft, which is thecurrent procedure in the repair of severe injuries of peri-pheral nerves. By using an arti"cial guide, the need toremove a healthy nerve segment is avoided and the lossof function in the donor site is prevented. However, it isnecessary to demonstrate that the arti"cial guide canensure a satisfactory nerve repair, achieving a degree ofregeneration and recovery at least as good as with anautologous graft. In a previous study we showed thatPLC impermeable guides allowed regeneration throughlonger gaps and with signi"cantly higher levels of rein-nervation than possible with synthetic silicone and te#on


Fig. 5. Evolution of the amplitude of (A) the gastrocnemius CMAP, (B)the plantar CMAP and (C) the digital CNAP over time in groups ofmice with sciatic nerve resection and repair with di!erent nerve guidesmade of poly(L-lactide-co-e-caprolactone) (PLC) or polysulfone (POS).

tubes [20]. The present study also shows that bioresorb-able PLC guides provide better conditions for nerveregeneration than durable POS guides. Moreover, byusing PLC tubes of high permeability regeneration andreinnervation was considerably improved with respect toimpermeable or low-permeable tubes.

Our results are in agreement with those of other re-ports, which showed that resorbable guides allow forbetter levels of regeneration than permanent ones[20}22]. The reasons for this improvement are still un-known, although it may be hypothesized that resorbabletubes allow for better nutrient supply to the regeneratednerve, enhance the constitution of the initial matrix andthe subsequent nerve cable, and increase their #exibilityas they degrade, thus avoiding secondary damage tothe maturing regenerated nerve. Complications derivedfrom mechanical damage to axons and demyelination

associated with tubulization with permanent guides, re-sulting in loss of regenerating "bers, neuroma formationand pain [5,6,23,24], have also increased the interest fordeveloping suitable bioresorbable materials. However,bioresorbable tubes that degrade too quickly may notprovide for a long enough time an adequate space fornerve regeneration and maturation. If the nerve guidebreaks down at an early stage, "brous tissue can beformed inside the tube and impair further maturation ofthe regenerated nerve [25]. Another disadvantage ofdegradable nerve guides may be that the cellular activityduring the process of degradation may cause deleteriouse!ects on the regenerated nerve [25]. Ideally, a nerveguide should be composed of a biocompatible, bioresorb-able material that degrades at a controlled rate in accord-ance with the rate of axonal growth and maturation,maintaining mechanical continuity and lumen stabilityfor longer time than required for the axons to cross thegap. The PLC guides that we implanted are well toler-ated by the host tissue, elicit only a mild foreign bodyreaction, and have a low resorption rate for over6 months [17,26], a period long enough for the regen-erated nerve to be well formed over a long gap.

Successful regeneration after tubulization repair de-pends on the formation of a new extracellular matrixsca!old, over which blood vessels, "broblasts and lateron Schwann cells migrate and form a new nerve structure[2,27]. Surviving axons in the proximal stump elongateinto the tube along cellular outgrowths that follow theconnective strands bridging the gap, and eventually theaxons and accompanying Schwann cells progress intothe distal nerve stump. This implies a delay in axonalelongation, and failure of regeneration if the nervestumps are not able to provide a proper cable withenough regenerative promoting elements inside the tube,as often occurs in long gaps [11,28,29]. Therefore, regen-eration fails through long gaps most likely because theregenerative capabilities of the nerve stumps have beenexceeded and "broblasts and Schwann cells are not ableto migrate through the gap and provide a permissiveenvironment for axonal elongation. In these situations,the inmigration through the tube wall of extraneuralwound-healing di!usable factors or of "broblasts andmacrophages may represent an additional support.

The temporal sequence of nerve regeneration in sili-cone tubes and in semipermeable acrylic tubes followsa similar pattern and produces regenerates that are sim-ilar in their overall cytoarchitecture [2,10,30]. Whenbridging short or medium length gaps, such as 4 mm inthe mouse and 4}8 mm in the rat, tubulization is usuallysuccessful, provided that the physical parameters of thenerve guide are adequate and the lumen is not occluded[29,31}33], and semipermeable tubes behave similar orslightly worse than impermeable silicone tubes [12,13].However, when the gap is longer than the maximallength that can be regenerated within silicone tubes, i.e.


Fig. 6. Micrographs of transverse sections of successfully regenerated nerves through nerve guides. (a) impermeable PLC, (b) highly permeable PLC,(c) low-permeable PLC and (d) 30 KDa semipermeable POS guides. Note the "ber grouping in small fascicles and the changes in "ber density andmyelin thickness. Final magni"cation is ]1000.

6 mm in the mouse and 12}15 mm in the rat, or the distalend is left without a nerve insert, semipermeable tubesimprove the regeneration success rate [12,30]. Aebischeret al. [12] found nerve cables containing myelinated andunmyelinated "bers that regenerated through blind-ended perm-selective 50 kDa POS tubes 6 mm long inthe mouse, but no axonal regenerates in blind-endedimpermeable tubes. They suggested that the ability of theguides to support nerve regeneration relied on its per-meability characteristics more than in its chemical com-position, as the same capped acrylic tubes rendered

impermeable supported no regeneration. Our previousresults showing that permeable collagen guides allowedsimilar levels of regeneration than silicone tubes througha 4 mm gap in the mouse, but successful regenerationthrough a 6 mm gap in more mice than silicone tubes[20,34], support the view that tube permeability, durabil-ity and composition in#uence the chances of regenera-tion through long gaps. In addition, the microgeometryof the guide internal surface has been shown to a!ectnerve regeneration. Acrylic tubes with a rough surfaceinduced the formation of a loose connective tissue stroma


Table 2Morphological parameters of the regenerated nerves at mid-tube and distal levels in the "ve groups of mice studied and in control sciatic nerves. Valuesare expressed as mean$SEM. All animals were included for nerve area and number of myelinated "bers, while only animals with a regenerated nerveinside the guide were included for axonal diameter and g ratio

Group Nerve area (mm2) Myelinated "bers (no.) Axonal diameter (lm) g ratio

n Mid Mid Distal n Mid Distal Mid Distal

Control 4 0.21$0.01 4522$166 3 3.45$0.06 0.62$0.01PLC-np 9 0.07$0.03! 1048$442! 987$436! 5 3.15$0.13 2.40$0.04!," 0.67$0.02" 0.65$0.01!

PLC-lp 6 0.11$0.07 2715$1536 218$171! 4 2.03$0.16!,# 2.15$0.14!,# 0.62$0.01 0.62$0.01#

PLC-hp 6 0.13$0.05 2869$1179 1971$688! 4 2.69$0.23 2.81$0.18 0.61$0.02 0.62$0.03POS-30 7 0.04$0.03! 874$789 198$131! 2 2.74$0.74 2.55$0.05 0.64$0.03 0.62$0.02POS-100 6 0.01$0.01! 163$136! 51$37! 2 2.57$0.64 2.13$0.06!,# 0.71$0.04 0.61$0.01#

P(0.05!vs. control."vs. PLC-hp.#vs. PLC-np.

that contained only a few regenerated axons in contrastwith the well-formed nerve cable with numerous axonsfound in smooth-walled tubes made of the same material[35]. The surface of the PLC guides, although not assmooth as that of POS or silicone tubes, did not exerta negative in#uence. Observations during "nal dissectionand histological assessment showed that the regeneratedcable was centered in the guide lumen and not adhered tothe wall, and the number of regenerated axons was con-siderably higher than in POS and silicone guides [20].

The degree of permeability seems to exert signi"cantin#uences upon the success of nerve regeneration, al-though it is also dependent on the gap length. Thus, thesize of the nerve cable and the number of regeneratedaxons was similar inside POS tubes of 100 and 1000 kDaMW cut-o!with a 4 mm gap in the hamster sciatic nerve,but when the gap was 8 mm the 1000 kDa tubes con-tained signi"cantly less axons than the 100 kDa ones[36]. These results suggested that semipermeable guidesallow for a controlled exchange between the wound-healing environment and the tube lumen of externalmolecules which can either promote or inhibit regenera-tion [36]. In our study, we also found better, though notsigni"cantly, regeneration in 30 kDa than in 100 kDaPOS tubes. However, the "nal levels of functional recov-ery for all functions tested were poor for both groups,similarly to those found by using impermeable tubes witha 6 mm gap in themouse sciatic nerve [20,29]. By makinglarge holes in the wall of silicone tubes, thus allowing theextraneural #uid and cells free access to the intratubularregenerating space, Jenq and Coggeshall [11,37] founda higher proportion of successful regeneration, higheraxon numbers in the tube and into the distal stump, anda longer gap between the stumps that the axons couldspan in comparison with impermeable tubes. This im-provement in regeneration was similar when the holeswere covered with "lters of 5 lm pore size, that allowed

both soluble substances and cells to exchange, but notwith "lters of 1.2 lm pore size that impeded extraneuralcells entrance into the tube [38]. They pointed out thatthe majority of cells entering the tube were most likely"broblasts and reticuloendothelial cells, that would con-tribute in the formation of an extracellular matrix thatpromote or support axonal growth.

The increased wall permeability of nerve guides shouldplay a role during the early phases of regeneration, whenthe initial connective cable is formed bridging the prox-imal and distal stumps. In a few weeks a "brous cover, incontinuity with the epineurium of the nerve stumps, isformed surrounding the guide and limiting the exchangebetween the intratubular and the outer milieu, and theperineural-nerve barrier is reestablished [39]. The laterprogressive increase in wall permeability of bioresorb-able tubes as the material degrades may be of lesserrelevance in enhancing axonal regeneration. On the con-trary, if degradation or permeability changes in the tubewall occur too fast, when the regenerated nerve cable isnot well matured and a perineurial-nerve barrier has notyet been reestablished, the regenerated axons may su!erfrom mechanical or biochemical damage [39]. Bio-resorbable impermeable guides composed of PLC haveshown to o!er good support for nerve regeneration[20,25,26,33]. Considering that they have the same chem-ical composition and similar biodegradation rate, theimproved results found with the PLC guides of high-permeability wall might be attributable to the fact that, inaddition to maintain good mechanical stability and a lowresorption rate, they allow for the entrance of moleculesand cells that promote nerve regeneration. It is worth tonote that if considering only the mice with successfulregeneration in the PLC-hp tubes, the results are similarto those found with a sciatic autograft of the same gaplength [34]. On the other hand, the PLC tubes of lowpermeability have the disadvantage of inducing the


formation of neuromata, likely due to their mechanicalinstability, and allow distal regeneration of considerablylower number of axons, that may be explained byan imbalance in the in#ow of growth promoting andinhibitory substances and the limitation to cellularingrowth.

Acknowledgements

The authors thank Amicon for providing polysulfonehollow "bers, and Kerr Co. for supplying Elasticon ma-terial. This work was supported by grants from the FIS(FIS95-0033-02) and from the CICYT (SAF97-0147),Spain.

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highly permeable polylactide-caprolactone nerve guides enhance peripheral nerve regeneration through...

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